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Planar Chromatography

Thin Layer Chromatography

The beginning: Paper chromatography

Deposition of
a drop of

Deposition of
a drop of

Deposition of
more solvent

Chromatography = Chroma (colour) + Graphein (Writing)
Chromatography = Colour Writing

The beginning: Paper chromatography .

Evolution: Vertical paper chromatography Sample is deposited at the bottom line of the paper Solvent migrates in the paper and elutes the solutes Paper is placed in a tank filled with 1 cm solvent The solute migrate depending on their affinity for the solvent .

alumina) deposited on a rectangular glass plate (10-20 cm large) Current evolution: High-Performance TLC (HPTLC) Controlled size of stationary phase particles Modified stationary phases (bonded silica: ODS. chiral…) Automated procedures for better reproducibility .Thin Layer chromatography (TLC) The modern version of paper chromatography Paper is replaced by a layer (100-200 μm) of stationary phase (silica gel.

Thin Layer chromatography (TLC) Sample deposition Sample volume: a few nL to a few μL Sample is preferably deposited using an automated apparatus .with a drying gas spraying the sample Deposit is thiner and more even Better resolutions can be achieved .in a band-shape .

Thin Layer chromatography (TLC) Use of concentration zone .

Thin Layer chromatography (TLC) Pre-saturation of the layer is often preferable Solvent is volatile Effect of pre-equilibrium of a TLC plate Allows pre-saturating the layer with solvent vapors prior to development Solvent front migrates less rapidly Better separations can be achieved .

Thin Layer chromatography (TLC) Vertical Development 1. Solvent in Liquid-Vapour equilibrium 2. Solvent in Vapour adsorbs on the layer 3. Solvent migrating in the layer vaporizes Effect of gravity Migration distance In presaturated chamber In non saturated chamber Analysis time .

reservoir for developing solvent 4. HPTLC plate (layer facing down) 2. glass plate for sandwich configuration 3. glass strip 5. conditioning tray No effect of gravity Migration speed is constant Better resolutions can be achieved .Thin Layer chromatography (TLC) Horizontal Development 1. cover plate 6.

Thin Layer chromatography (TLC) Horizontal Development Better control of the operating conditions (saturation. evaporation) Possibility to develop both sides of the plate = Twice more samples .

Thin Layer chromatography (TLC) 2D separation Spotting the plate 1st elution 90° rotation 2nd elution Different mobile phases = different principles of separation Analogy: 2D-gel electrophoresis used in biotechnology .

Thin Layer chromatography (TLC) Reading the TLC Totally unretained solute Solvent front d solute Rf  d solvent dsolvent dsolute Starting line Totally retained solute .

Thin Layer chromatography (TLC) Detection of the analytes Coloured analytes Derivatisation procedures Densitometry with UV scanner UV light beam Detector Reflected beam Pseudo-chromatogram .

Thin Layer chromatography (TLC) Detection of the analytes Absorption of UV radiation is proportional to concentration Quantification is possible .

Thin Layer chromatography (TLC) Detection of the analytes Mass spectrometry (Analogous to Matrix Assisted Laser Desorption Ionisation) Laser Desorption Desolvation  + + H+ Proton transfer   .

Thin Layer chromatography (TLC) Advantages: Drawbacks: Easy to use Cheap Possible multiple analysis Possible recovery of the products No sample preparation required 2-dimensional analysis Slow (typically 30-60 minutes) Limited quality of the separation Limited reproducibility Evaporation of the mobile phase (composition varies during the analysis) .

4°C UV-densitometry at 275 nm Vanhaelen-Fastré et al. Chromatogr.8 (v/v/v/v).2:4.8:19. J.2-dichloroethane – ethanol – methanol – water 56.2:19..Example: ginsenoside solutes ginsenosides standards solution Triterpene glycosides Used in traditional asian medicine and occidental phytotherapy extract of Panax ginseng Silica gel 1. 868 (2006) 269-276 . A.

A. antifungal. 2.Example: anthraquinone derivatives 4 major anthraquinone derivatives found in a species of indian rhubarb Varied bioactivities (antioxidant. H H H Glc OCH3 H OH H Physcion Chrysophanol Emodin Chrysophanol Glycoside RP-18 thin layer methanol – water – formic acid 80:19:1 (v/v/v) UV-densitometry at 445 nm Singh et al. 4. antiviral. etc) Compound R1 R2 1. antimicrobial. J. Chromatogr. 3.. 1077 (2005) 202-206 .